Method of evaluating the extent of reaching the intestine of bifidobacterium in fermented milk food or drink

ABSTRACT

A  Bifidobacterium  that is resistant to acid and bile and which retains at least 20% viability after acid and bile treatment. Foods and drinks containing an acid- and bile-resistant  Bifidobacterium . Methods for providing viable  Bifidobacteria  to the intestine after ingestion of a food or drink containing an acid- and bile-resistant  Bifidobacterium.

This application is a national-stage filing under 35 U.S.C. §371 ofPCT/JP01/11266, filed Dec. 21, 2001. It also claims priority to JAPAN2001-25297, filed Feb. 1, 2001.

TECHNICAL FIELD

The present invention relates a convenient method for assessing theextent to which Bifidobacterium bacteria in a fermented milk food ordrink reach the intestines when the food or drink is administered orallyto human beings; and Bifidobacterium bacteria having a high viabilitywhen assessed by the method.

BACKGROUND ART

Bifidobacterium bacteria are reported to exhibit various physiologicaleffects such as inhibitory effects against harmful intestinal bacteria,intestinal function controlling effects and immunoactivating effects. Anumber of products containing Bifidobacterium bacteria have been put onthe market, for example, in the form of fermented milk products orlive-bacteria-containing preparations, and they have established a firmposition in the market. In particular, a strong preference to fermentedmilk food and drink enables continuous intake of Bifidobacteriumbacteria and they are therefore a suitable form for administration.

Bifidobacterium bacteria exhibit most of their physiological effects byproducing acetic acid or the like as a metabolite after reaching theintestines. In order to produce satisfactory effects, they must reachthe intestines in the viable form. The extent to which bacteria reachthe intestines has conventionally been judged with their recovery ratefrom feces as an index. Described specifically, the number of livingbacteria in the feces reflects the extent to which the bacteria reachthe intestines so that the extent has been confirmed by collecting thefeces of the human beings to which the bacteria were administered andcounting the number of living bacteria (collection rate) in the feces.

It is generally considered that Bifidobacterium bacteria have difficultyin reaching the intestinal tracts because of weak tolerance to an acidand bile. In other words, when they are taken orally, there is apossibility of them being killed by gastric juice or bile prior toreaching the intestines.

In recent years, improvement of various production techniques enables anincrease in the very number of bacteria which can be administered sothat even if some of the bacteria are killed, physiological effects canstill be expected from the remaining ones. Upon production of fermentedfoods containing Bifidobacterium bacteria, a variety of components forimproving bacterial viability during storage, for example, N-acetylglucosamine, pantothenic acid, peptides and lactulose are added. Theiraction is considered to heighten the extent to which Bifidobacteriumbacteria reach the intestines after their administration.

In order to attain stronger physiological effects, a greater number ofviable bacteria must be caused to reach the intestines. There isaccordingly a demand for improving the extent to which the bacteriareaching the intestines. An administration test to human beings becomesinevitable for finding the extent, that is, a recovery ratio of themfrom feces. Cumbersome and long-term works necessary for the test havehowever hampered the implementation of such a test.

There are some reports on the production of a strain endowed in advancewith tolerance to gastric juice or bile which will otherwise be abarrier against the orally taken bacteria. For example, it is describedin Japanese Patent Laid-Open No. Hei 9-322762 that use ofBifidobacterium bacteria exhibiting high tolerance to acids, bile saltand oxygen enables culture on skim milk or the like under aerobicconditions without adding a growth promoting substance. In InternationalJournal of Food Microbiology, 47, 25-32(1999), described are two strainsof Bifidobacterium bacteria having high tolerance to an acid and bile.

Considering the possibility of improving the extent to which bacteriareach the intestines upon their administration by the use of such highlytolerant bacteria, the present inventors created such a strain havinghigh tolerance to an acid and bile, prepared a syrup-containingfermented milk food or drink by using the strain, administered it tohuman beings and found a recover ratio from feces. As a result, it hasbeen revealed that there is not always a correlation between thetolerance and the recovery ratio.

Accordingly, an object of the present invention is to construct aconvenient and accurate assessing system which reflects the extent towhich Bifidobacterium bacteria in a fermented milk food or drink reachthe intestines. Another object of the present invention is to create astrain superior in the extent to the conventional Bifidobacteriumbacteria and provide a Bifidobacterium-bacteria-containing fermentedmilk food or drink by using the bacteria.

DISCLOSURE OF THE INVENTION

The present inventors carried out an investigation on a recovery ratioof Bifidobacterium bacteria from feces of persons to whom aBifidobacterium-bacteria-containing fermented milk food or drink hadbeen administered. As a result, it has been found that in a fermentedmilk food or drink, particularly, a syrup-containing fermented milk foodor drink, a recovery ratio is relatively high just after thepreparation, but it shows a drastic reduction after about one week fromthe preparation. When the distribution of fermented milk food and drinkis taken into consideration, a sufficiently high recovery ratio must bekept even after storage for such a period. The present inventors havecarried out a further investigation using a Bifidobacterium-bacteria-and syrup-containing fermented milk food or drink stored for about oneweek. As a result, it has been found that the extent to whichBifidobacterium bacteria in a fermented milk food or drink reach theintestines when the food or drink is orally administered can be assessedconveniently and accurately by measuring bacterial viability aftersuccessive treatments with an acid and bile. It has also been found thatwhen a fermented milk or food containing a strain which exhibits atleast predetermined bacterial viability, with the bacterial viabilityafter successive treatments with an acid and bile as an index, isadministered, the extent to which bacteria reach the intestines isexcellent even about one week after the preparation of the food ordrink, leading to completion of the invention.

In one aspect of the present invention, there is thus provided a methodfor assessing the extent to which Bifidobacterium bacteria in aBifidobacterium-bacteria-containing fermented milk food or drink reachthe intestines when the fermented food or milk is orally administered tohuman beings, which comprises successively treating the fermented milkfood or drink with an acid and then bile; and measuring bacterialviability after these treatments.

In another aspect of the present invention, there are also providedBifidobacterium bacteria exhibiting at least 20% of bacterial viabilityas measured by the above-described assessing method; and aBifidobacterium-bacteria-containing fermented milk food or drink, whichcomprises the above-described bacteria.

BEST MODE FOR CARRYING OUT THE INVENTION

Any Bifidobacterium-bacteria-containing fermented milk food or drink isusable for the assessment in the present invention. Examples includefermented milk food and drink each obtained by adding a syrup to afermented milk prepared by culturing Bifidobacterium bacteria on anutrient medium such as GAM medium (product of Nissui Pharmaceutical)and then culturing the resulting bacteria as a seed on a milk medium. Inparticular, the extent to which Bifidobacterium bacteria in asyrup-containing fermented milk food or drink reaches the intestineslowers during storage so that the assessing method of the presentinvention exhibits effectiveness for syrup-containing fermented milkproducts. The term “milk” as used herein 4means cow milk (whole milk),skim milk which is a processed product thereof, milk of another animalsuch as goat, sheep or the like, and soy milk which is of plant origin.Examples of the syrup include saccharides such as glucose, sucrose,fructose-glucose syrup, glucose-fructose syrup and comb honey; and sugaralcohols such as sorbitol, xylitol, erythritol, lactitol, and palatinit.The content of the syrup is preferably 0.1 to 10 wt. %. The fermentedmilk food or drink preferably contains 1×10⁷ to 5×10⁹ cfu/mL,particularly preferably 1×10⁸ to 1×10⁹ cfu/mL of Bifidobacteriumbacteria. The number of bacteria exceeding 5×10⁹ disturbs accuratejudgement, because the burden of an acid or bile is insufficient forsuch a count. On the other hand, the number of bacteria less than 1×10⁷is inappropriate, because the burden of an acid or bile on each bacteriais excessively large.

In the fermented milk food or drink, an emulsifier such as sucrose fattyacid ester, polyglycerin fatty acid ester or lecithin, and a thickener(stabilizer) such as agar, gelatin, carrageenan, guar gum, xanthan gum,pectin or locust bean gum can be incorporated. Vitamins such as vitaminA, B-vitamins, vitamin C and vitamin E, and herb extracts can also beincorporated.

Microorganisms other than Bifidobacterium bacteria can be used incombination therewith for the preparation of a fermented milk food ordrink. Preferred examples of microorganisms include Lactobacillusbacteria such as Lactobacillus acidophilus, Lactobacillus gasseri,Lactobacillus plantarum, Lactobacillus buchneri, Lactobacillus casei,Lactobacillus johnsonii, Lactobacillus gallinarum, Lactobacillusamylovorus, Lactobacillus brevis, Lactobacillus rhamnosus, Lactobacilluskefir, Lactobacillus paracasei, Lactobacillus crispatus andLactobacillus delbrueckii subsp. delbrueckii; Streptococcus bacteriasuch as Streptococcus thermophilus; Lactococcus bacteria such asLactococcus lactis subsp. lactis; Bacillus bacteria such as Bacillussubtilis; and yeasts belonging to the genera Saccharomyces, Torulasporaand Candida such as Saccharomyces cerevisiae, Torulaspora delbrueckiiand Candida kefir.

A fermented milk food or drink prepared using, in combination withBifidobacterium bacteria, at least one lactic acid bacteria selectedfrom the above-described Lactobacillus, Streptococcus and Lactococcusbacteria is preferred, because owing to a high preference to such adrink, it facilitates continuous administration and in addition, it ishighly effective for improving the intestinal reaching degree orrecovery ratio.

The fermented milk food or drink may be prepared in a manner known perse in the art. For example, a fermented milk base is prepared byinoculating and culturing Bifidobacterium bacteria singly orsimultaneously with lactic acid bacteria on a sterilized milk medium,and then homogenizing the cultured bacteria. To the resulting fermentedmilk base is added a syrup solution which has been prepared separately.After mixing, the mixture is homogenized in a homogenizer, followed bythe addition of a flavor, whereby a final product is prepared.

The fermented milk food or drink thus available can be provided in anyone of soft type, fruits flavor type, solid form and liquid form.

The fermented milk food or drink to be subjected to successivetreatments with an acid and bile is preferably that which has beenstored at low temperatures for 7 days after completion of theincubation, with the storage starting day reckoned as 0 (in the case ofa syrup-containing fermented milk food or drink, with the day on whichthe syrup and Bifidobacterium bacteria were added reckoned as 0). It haspreferably a pH of from 5.0 to 5.8.

Although no particular limitation is imposed on the strain of theBifidobacterium bacteria to be employed, those derived from human beingsare preferred. Preferred are Bifidobacterium breve, Bifidobacteriumbifidum, Bifidobacterium longum, Bifidobacterium infantis,Bifidobacterium adolescentis, Bifidobacterium catenulatum andBifidobacterium pseudocatenulatum, of which Bifidobacterium breve,Bifidobacterium bifidum and, Bifidobacterium iongum are particularlypreferred.

The term “successive treatments with an acid and bile” as used hereinmeans treatment of the fermented milk food or drink with an acid,followed by treatment with bile. The term “successive treatments” asused herein means that the treatment with bile is performed instantlyafter the treatment with an acid or performed without recovering theactivity of bacteria. The term “without recovering the activity ofbacteria” means that no positive treatment is given for recovering theactivity of bacteria, for example, addition of a neutralizer to the acidtreated solution or dilution with a buffer or medium.

The term “acid treatment” as used herein means treatment of adding thefermented milk food or drink to a solution, which has been made moreacidic than the optimum pH of Bifidobacterium bacteria by the additionof an organic acid such as acetic acid, lactic acid, citric acid, malicacid, succinic acid, butyric acid or propionic acid, or an inorganicacid such as hydrochloric acid, sulfuric acid, nitric acid or phosphoricacid, thereby causing damage to the food. Although no particularlimitation is imposed on the acid to be employed, use of an organic acidis preferred, because existence of a salt adversely affects the growthof some strains of Bifidobacterium bacteria and this influence cannotalways be neglected when a correlation between bacterial viability and arecovery ratio is considered. Use of acetic acid, lactic acid orhydrochloric acid heightens a correlation between bacterial viabilityand the extent to which bacteria reach the intestines and is thereforeparticularly preferred.

The acid containing solution to be used for the acid treatment ispreferably adjusted at pH of from about 3.0 to 5.5, especiallypreferably from about 4.0 to 4.5. When the pH exceeds 5.5, it becomes,in most cases, higher than that of popular fermented milk productsthemselves and can cause only a slight damage to the Bifidobacteriumbacteria. At pH less than 3.0, on the other hand, most of the bacteriawill be killed by such a strong acid solution within an extremely shorttime. Thus, at pH outside the above-described range, a differencebetween strains in the bacterial viability after the treatment cannot bedetected.

After the addition of the fermented milk food or drink to theacid-containing solution, the resulting mixture may be treated for about1 to 60 minutes at a temperature suited for the growth ofBifidobacterium bacteria, that is, about 30 to 42° C. Treatment at about36 to 38° C. for about 10 to 30 minutes is preferred, for suchconditions reflect the living conditions of the bacteria and areconvenient for the implementation of the test.

In the present invention, the fermented milk food or drink after theacid treatment is treated with bile. Described specifically, thefermented milk food or drink after the acid treatment is added directlyto a bile solution. Although no particular limitation is imposed on theconditions of the treatment with bile, it is recommended to add theacid-treated fermented milk food or drink to a solution obtained byadding sodium phosphate as a neutralizer to about 0.1 to 5% of bile orbile salt, and treat the mixture at 30 to 42° C. for about 1 to 240minutes. In order to heighten the correlation between the bacterialviability and the extent to which bacteria reach the intestines andclearly differentiate the tolerance between strains, it is preferred toadd about 0.5 to 2% of bile or bile salt and treat the mixture at 36 to38° C. for about 30 to 60 minutes.

More preferable successive treatments with an acid and bile aretreatments of a fermented milk food or drink, which containsBifidobacterium bacteria (the original number of bacteria: about 1×10⁸)and has been stored for 7 days, with an acid of pH 4.3 at 36 to 38° C.for 10 to 30 minutes and then with 1% bile at 36 to 38° C. for 30 to 60minutes.

Particularly preferable specific conditions of the successive treatmentswith an acid and bile are as follows: First, 1 mL of a fermented milkfood or drink is added to 10 mL of an acid treatment solution adjustedwith acetic acid to pH 4.3 and then, the resulting mixture is treated at37° C. for 30 minutes. Rightly after the first treatment, 1 mL of theacid treated solution is added to 10 mL of a bile treatment solutioncontaining 1% of bile, followed by treatment therewith at 37° C. for 30minutes. The treatment under such conditions enables the bacterialviability to roughly coincide with the extent to which bacteria reachthe intestines irrespective of the kind of the strains to be assessed.They also make it possible to clearly differentiate the bacterialviability between strains.

The bacterial viability after the successive treatments with an acid andbile can be determined by comparing the number of bacteria in thefermented milk food or drink before the treatments with that after thetreatments. In other words, a ratio of the number of bacteria after thetreatments to that before the treatments may be calculated. The numberof bacteria can be measured in a manner known per se in the art.

The bacterial viability of Bifidobacterium bacteria after suchsuccessive treatments with an acid and bile shows a close correlationwith the extent to which Bifidobacterium bacteria in a fermented milkfood or drink reach the intestines when the milk or food is orallyadministered to human beings. Described specifically, the bacterialviability after the successive treatments serves as a parameter whichvaries depending on the extent to which Bifidobacterium bacteria reachthe intestines upon oral administration of the fermented milk food ordrink to human beings. It is not known exactly but this closecorrelation is considered to owe to the fact that the stress applied toBifidobacterium bacteria, first by the acid treatment to lower theiractivity and then by the treatment with bile, is analogous to the stressoccurring in the human intestinal tracts.

The term “extent to which bacteria reach the intestines” as used hereinmeans, in the case where a Bifidobacterium-bacteria- andsyrup-containing fermented milk food or drink is orally administered tohuman beings, a ratio of the number of the bacteria reaching theintestines to the number of administered bacteria. The number ofbacteria reaching the intestines is considered to be almost similar tothe number of bacteria recovered from the feces so that in practice, thelatter number is regarded as the number of bacteria reaching theintestines.

Even if the bacterial viability is measured after either one of the acidtreatment or the bile treatment, no correlation is available between thebacterial viability and the extent to which bacteria reach theintestines or recovery ratio. When the acid treatment and bile treatmentare conducted at some interval, or when the bile treatment is followedby the acid treatment, bacterial viability has no correlation with theextent to which bacteria reach the intestines or recovery ratio.

When the conventional Bifidobacterium bacteria were employed, forexample, for food or drink such as fermented milk product, the extent towhich bacteria reach the intestines or recovery ratio of them wassufficient when the product was taken just after preparation, but itlowered when the product was supplied after about one weekrefrigeration. This tendency was marked in a syrup-containing fermentedmilk food or drink. The strain which has been confirmed to have highbacterial viability by the assessing method of the present invention isexcellent in the extent to which bacteria reach the intestines orrecovery ratio even after one week storage in the form of a fermentedmilk food or drink.

In the present invention, it has been confirmed that a fermented milkfood or drink, which has been prepared using a novel strain havingbacterial viability of 20% or greater, more preferably, 30% or greaterafter the successive treatments with an acid and bile, attains a similarlevel of the extent to which bacteria reach the intestines or recoveryratio to that just after preparation even if it is stored for about 1week. In spite of the presumption that the higher the number ofBifidobacterium bacteria in a fermented milk food or drink, the higherthe extent to which the bacteria reach the intestines, the results ofthe study by the present inventors have revealed that the extent tendsto lower when the food is stored for 1 week, resulting in a decrease inthe recovery rate from feces.

When a fermented milk food or drink containing at least 1×10 ⁸ cfu/ml ofhuman-derived Bifidobacterium bacteria exhibits bacterial viability of20% or greater after refrigeration for one week, the treatment at 37° C.for 30 minutes with an acid treatment solution adjusted to pH 4.3 with10 times the weight of acetic acid and then, the treatment at 37° C. for30 minutes with 10 times the weight of 1% bile acid, the number ofbacteria reaching the intestines can be kept at about 1×10⁷ cfu/g whenthe food or drink is administered to human beings after one weekstorage. Fermented milk food and drink equipped with such number ofbacteria and bacterial viability are particularly preferred, because ifthe number of bacteria reaching the intestines can be kept at about1×10⁷ cfu/g, proliferation of Bacteroides spp. and Clostridium spp.bacteria which are called “intestinal bad bacteria” can be controlled.

There is no particular limitation imposed on the strain ofBifidobacterium bacteria having high bacterial viability after thesuccessive treatments with an acid and bile, and a high extent to whichbacteria reach the intestines or high recovery ratio can be expectedfrom the use of any human-derived strain exemplified above. Among them,Bifidobacterium breve, Bifidobacterium bifidum and Bifidobacteriumlongum are preferred, because physiological effects and safety forinfants or the aged have already been confirmed well.

Strains having high tolerance to such successive treatments with an acidand bile can be produced, for example, by the following process.

To a fermented milk prepared using Bifidobacterium bacteria as a parentstrain, a syrup is added to prepare a fermented drink. The drink isdispensed into an appropriate container and it is refrigerated. Thefermented drink after refrigeration is subjected to acid and biletreatments, followed by centrifugal separation to recover the bacteria.From these bacteria, those having excellent tolerance to successivetreatments with an acid and bile are selected.

Although any strain of Bifidobacterium bacteria is usable for theabove-described method without limitation, but bacteria selected fromBifidobacterium breve, Bifidobacterium bifidum and Bifidobacteriumlongum are preferably employed because of the same reasons as describedabove.

Two strains YIT 4125 and YIT 4126 which have been recognized to havehigh bacterial viability after the successive treatments with an acidand bile as a result of the above-described method are deposited withInternational Patent Organism Depositary, National Institute of AdvancedIndustrial Science and Technology (Chuo 6 1-1-1 Higashi, Tsukuba,Ibaraki, Japan) as of Oct. 27, 2000; the former as Bifidobacterium breveYIT 4125 (FERM BP-7813) and the latter as Bifidobacterium breve YIT 4126(FERM BP-7814).

Alternatively, a strain having high bacterial viability after thesuccessive treatments with an acid and bile can be selected by exposingit to ultraviolet rays or treating it with a mutagen such asnitrosoguanidine (NTG) or ethylmethane sulfonate (EMS).

The Bifidobacterium bacteria having high bacterial viability after thesuccessive treatments with an acid and bile, which bacteria have beenobtained by the above-described method or another method, exhibit a highextent to which bacteria reach the intestines or recovery ratio. Theabove-described two strains are excellent strains with bacterialviability of 35% or greater after the successive treatments with an acidand bile.

In addition, the present invention relates to Bifidobacterium-bacteria-and syrup-containing fermented milk food and drink having high bacterialviability after the successive treatments with an acid and bile. Theforms of the fermented milk food and drink are similar to thosedescribed above.

EXAMPLES

The present invention will hereinafter be described in further detail byExamples. It should however be borne in mind that the present inventionis not limited to or by them.

Referential Example 1

The bacteria obtained by treating the Bifidobacterium Breve 4052 strain(B. breve A), a parent strain, with N-methyl-N′-nitrosoguanidine (NTG)were cultured on an anaerobic skim milk medium. To 10 mL of an acidtreatment solution (pH 3.8) was added 1 mL of the resulting culturesolution, followed by treatment at 37° C. for 8 minutes. On an anaerobicskim milk medium, 1% of the resulting solution was inoculated andcultured at 37° C. for 20 hours. After storage of the culture solutionat 10° C. for 4 days, 1 mL of the resulting solution was added to 10 mLof an acid treatment solution (pH 3.8), whereby a similar treatment wasconducted. The bacteria after the treatment were cultured on a skim milkmedium. Several single-colony derived strains were selected from theculture and their acid tolerance was compared. As a result, theBifidobacterium breve N4 strain (B. breve B) was selected as a strainhaving improved acid tolerance. The strain had improved bile toleranceat the same time.

Composition of Anaerobic Skim Milk Medium

Skim milk 12%  Yeast extract 1% Cysteine   0.03% Calcium carbonate 2%

Nitrogen was blown into the medium for 20 minutes, followed by sealingwith a rubber stopper and autoclave sterilization at 115° C. for 20minutes.

Example 1

Three fermented milks were prepared by inoculating seed cultures of B.breve A, B. breve B (N4 strain) and B. bifidum C (YIT 4007 strain) on a18% whole milk medium (added with 0.03% yeast extract) sterilized at100° C. for 90 minutes, and culturing at 34° C. for about 20 hours. Toeach of the fermented milks thus obtained, a glucose-fructose syrup wasadded to give a final concentration of 8% and the resulting mixtureswere used as test samples. These samples were each dispensed in a glasscontainer. After the container was sealed with a rubber stopper to shutoff the air flow into the container, it was stored at 10° C. for 7 days.

Recovery of bifidobacteria from the feces of human beings administeredwith these samples was studied. Eighteen healthy adult male volunteers(average age: 33) were divided into three groups at random and they wereasked to drink, for 3 days, 100 mL/day of the fermented milk preparedusing each strain. On the next day after completion of drinking, feceswere recovered from them and diluted stepwise with an anaerobic dilutingsolution. From the number of colonies appearing on a selective plate,the number of recovered bacteria was calculated. A T-CBPC plate was usedfor B. breve A and B strains, while a T-LCM plate was used for the B.bifidum C strain.

Composition of Anaerobic Dilution Water

Potassium dihydrogen phosphate 0.0225% Dipotassium hydrogen phosphate0.0225% Sodium chloride 0.045% Ammonium sulfate 0.0225% Calcium chloride0.00225% Magnesium sulfate 0.00225%

After the above-described composition was dissolved in distilled water,nitrogen was brown into the resulting solution to make it anaerobic.With a rubber stopper, air was shut off, followed by autoclavesterilization (121° C. for 15 minutes).

Composition of T-CBPC Plate

TOS (product of Yakult) 1%   Trypticase peptone (BBL) 1%   Yeast extract(Difco) 0.1% Potassium dihydrogen phosphate 0.3% Dipotassium hydrogenphosphate  0.45% Ammonium sulfate 0.3% Magnesium sulfate  0.02% Cysteinehydrochloride  0.05% Lab-Lemco powder (OXOID) 0.1% Agar (Difco) 1.5%

The above-described composition was dissolved in distilled water,followed by autoclave sterilization at 115° C. for 15 minutes. Aftercooling to 50° C., Carbenicillin (Sigma) which had been sterile-filteredand Streptomycin sulfate (Sigma) were added to the resulting solution togive final concentrations of 1 μg/mL and 0.5%, respectively. An agarplate was prepared in such a manner.

Preparation of T-LCM Plate

A T-LCM plate was prepared in a similar manner to that employed for thepreparation of the above T-CBPC plate except that 2 μg/mL of Lincomycin(Sigma) was added instead of 1 μg/mL of Carbnenicillin (Sigma) as asterile-filtered antibiotic.

As a result, a difference in recovering property as described below wasobserved among three fermented milks prepared using three bifidobacteriastrains.

TABLE 1 Strain A B C The initial number of bacteria 3.99 × 10⁸ 3.16 ×10⁸ 7.94 × 10⁸ The number of bacteria recovered 4.79 × 10⁴ 4.37 × 10⁵9.55 × 10⁶

Example 2

In vitro model system which reflects the recovery property in Example 1was studied. With preference on convenience, the system permittingcompletion of treatment within from 30 minutes to 1 hour was studied.

First, tolerance to acid treatment was evaluated as a model of thestomach through which food passes first among digestive tracts. As anacid treatment solution, a solution having a pH lowered by the additionof an acid to a bifidobacteria medium was used, with reference to themethod of Kobayashi, et al. (Japanese Journal of Bacteriology, 29,691-697(1974)).

Composition of Acid Treatment Solution

Trypticase peptone (BBL) 1%   Yeast extract (Difco) 0.5% Tryptose(Difco) 0.3% Sodium chloride 0.2% Monoammonium citrate 0.2% Cysteinehydrochloride  0.05% Lactose 1%   Pyruvic acid 0.1% Tween 80 0.1%Magnesium sulfate   0.0575% Ferrous sulfate   0.0034% Manganese sulfate 0.012%

The above-described composition was dissolved in distilled water. Anacid such as acetic acid was added to adjust the pH of the solution to 3to 5.5, followed by autoclave sterilization at 121° C. for 15 minutes.

As a result of investigation on the pH of the acid treatment solution,it was found that the pH set at 3.8 or greater enabled measurement ofthe number of surviving bacteria after treatment for 1 to 30 minutes.The acid treatment solution having a pH of 3.8 was therefore employed.

To 10 mL of the acid treatment solution having a pH 3.8, 1 mL of each ofthe three samples used in Example 1 was added, followed by incubation at37° C. During the treatment, sampling was conducted as needed and thenumber of living bacteria was counted. The death velocity was determinedbased on the data thus obtained. The results are as shown below.

TABLE 2 Strain A B C Death velocity during acid treatment (logarithm of0.55 0.38 0.51 the number of bacteria killed per minute)

The above-described results show that the strain B is excellent in thedeath speed by the acid treatment, which, however, does not coincidewith the order of Example 1 in the number of bacteria recovered afteractual administration.

Example 3

In the next place, tolerance to bile treatment was compared assumingthat the bacteria reached the intestines. As a treatment solution, thathaving the below-described composition and added with Oxgall was judgedappropriate, when the treatment time and measurability of the number ofbacteria were taken into consideration as in the case of the acidtreatment.

Composition of Bile Treatment Solution

Trypticase peptone (BBL) 1%   Yeast extract (Difco) 0.5% Triptose(Difco) 0.3% Disodium hydrogen phosphate 12 hydrate  2.03% Sodiumdihydrogen phosphate 2 hydrate  0.156% Diammonium citrate 0.2% Cysteinehydrochloride  0.05% Lactose 1%   Pyruvic acid 0.1% Tween 80 0.1%Magnesium sulfate   0.0575% Ferrous sulfate   0.0034% Manganese sulfate 0.012%

The above-described composition was dissolved in distilled water. To theresulting solution was added 0.1 to 5% of Oxgall (Difco) to dissolve thelatter. in the former. The solution was adjusted to pH 8.0 with a sodiumhydroxide solution, followed by autoclave sterilization at 121° C. for15 minutes.

As a result of investigation on the concentration of Oxgall, it wasfound that the Oxgall concentration set at 1% or less enabledmeasurement of the number of surviving bacteria after the treatment for1 to 30 minutes. The bile treatment solution having an Oxgallconcentration of 1% was therefore employed. To 10 mL of the biletreatment solution containing 1% Oxgall, 1 mL of each of the threesamples used in Example 1 was added, followed by treatment at 37° C. for30 minutes. After the treatment, the number of living bacteria wascounted. The bacterial viability after the bile treatment was measuredbased on the data thus obtained. The results are as shown below.

TABLE 3 Strain A B C Bacterial viability (%) after bile treatment <0.160 30

It has been found that the strain B exhibited the highest tolerance tobile, which was however different from the actual recovery results.

Example 4

Also in vitro, acid treatment and bile treatment were conductedsuccessively in consideration that when the food passes through thedigestive tracts, it passes successively through the stomach andintestines. In these successive treatments with an acid and bile,adjustment of an acid treatment solution to pH 4.3 or greater and a biletreatment solution to have an Oxgall concentration of 1% enabledmeasurement of the number of surviving bacteria after treatment.Described specifically, the successive treatments with an acid and bileas described below were performed.

To 10 mL of an acid treatment solution adjusted to pH 4.3 with aceticacid was added 1 mL of the syrup-containing fermented milk as employedin Example 1, followed by treatment at 37° C. for 30 minutes.Immediately after the treatment, 1 mL of the acid treated solution wasadded to a bile treatment solution containing 1% of Oxgall, followed bytreatment at 37° C. for 30 minutes. A survival ratio of bacteria afterthe successive treatments with an acid and bile was compared among threestrains.

TABLE 4 Strain A B C Survival ratio (%) after successive treatments <1 310

According to the assessment results, the bacterial viability after thesuccessive treatments coincided with the actual recovery results ofExample 1. In other words, the survival ratio of the strain B was thehighest in the case of single treatment with an acid or bile, while thatof the strain C was the highest after the successive treatments with anacid and bile, which coincided with the results of the actual recovery.This suggests that recovery of bacteria administered through a drink canbe forecast based on the results of the in vitro successive treatments.

Example 5

The assessment by the above-described successive treatments wasperformed using a bred strain B. breve NE strain (strain D) to whichbile tolerance had been imparted by the below-described method.

First, the B. breve A strain used as a base was subjected to mutation. A50 mM phosphate buffer (pH 7.0) containing 5 μg/mL of NTG and the washedbacteria of B. breve were mixed at a ratio of 1:1. After treatment at37° C. for 30 minutes, the mixture was washed twice with a phosphatebuffer (pH 7.0). A nutrient medium was then inoculated with 1% of theresulting bacteria, followed by incubation at 37° C. for 16 hours.

The resulting NTG-treated bacteria were subjected to EMS treatment. A 50mM phosphate buffer (pH 7.0) containing 0.4% of EMS and theabove-described cultured bacteria were mixed at a ratio of 1:1. Aftertreatment at 37° C. for 30 minutes, the mixture was washed twice with aphosphate buffer (pH 7.0). A nutrient medium was inoculated with 1% ofthe resulting bacteria, followed by culturing at 37° C. for 16 hours.

By using the resulting bacteria as an initial seed (added with 0.03%yeast extract), a seed culture was prepared. It was inoculated into a18% whole milk medium sterilized at 100° C. for 90 minutes and culturedat 34° C. for about 20 hours, whereby a fermented milk was prepared. Aglucose-fructose syrup was added to the milk to give a finalconcentration of 8%. The resulting mixture was dispensed into a glasscontainer. After sealing with a rubber stopper, the container was storedat 10° C. for 10 days. Then, 10 mL of the sample after storage was addedto 100 mL of a bile treatment solution having the composition asdescribed in Example 4 and containing 2% of Oxgall. After bile treatmentat 37° C. for 30 minutes, the solution was centrifuged at 5000×g for 10minutes to recover the bacteria. The resulting bacteria were smeared onan agar plate. From the culture thus obtained, severalsingle-colony-derived strains were selected and the strain D havingimproved tolerance were obtained.

By using the resulting strain and the strain C, syrup-containingfermented milks were prepared as in Example 1. In a similar manner tothat employed in Example 4, the tolerance to successive treatments withan acid and bile was compared between these samples which had beenstored at 10° C. for 7 days. The results are shown below.

TABLE 5 (cfu/mL) Strain D C The original number of bacteria 2.1 × 10⁸5.3 × 10⁸ The number of surviving bacteria after 3.7 × 10⁷ 9.3 × 10⁷successive treatments Survival ratio 18% 18%

At the same time, drinking test was performed on these samples. Twelvehealthy adult volunteers were divided into two groups and were asked totake 100 mL/day of each of the two syrup-containing fermented milksprepared in Example 5 for 3 days. On the next day after completion ofthe drinking, feces were collected and diluted stepwise with ananaerobic dilution water. From the number of colonies appearing on theselective plate, the number of bacteria thus recovered was calculated. AT-CBPC plate was used for B. breve D strain, while a T-LCM plate wasused for B. bifidum C strain. The results are shown below.

TABLE 6 Strain D C The number of bacteria recovered (logarithm) 6.266.44

It has been found from these results that the strain D bred whilesubjecting it to single treatment with bile exhibited a survival ratioless than 20% after successive treatments with an acid and bile and thenumber of bacteria recovered after administration through drinking didnot reach 1×10⁷.

Example 6

Since it has been found that there is a high possibility that toleranceassessed by means of the successive treatments correlates with recoveryof bacteria after administration through drinking, a strain was bredwhile subjecting it to the successive treatments and the resultingstrain having tolerance was studied as to recovery of bacteria afteradministration through drinking.

A seed culture prepared using the strain A as a base was inoculated intoa 20% whole milk medium (added with 0.03% yeast extract) UHT-sterilizedat 135° C. for 3.5 seconds, followed by incubation at 34° C. for about20 hours, whereby a fermented milk was obtained. Palatinose was thenadded to the milk to give a final concentration of 10%. The resultingmixture was dispensed in a glass container. While the container wassealed with a rubber stopper to block the air from entering thecontainer, it was stored at 10° C. for 11 days. To 10 mL of the sampleafter storage was added 100 mL of an acid treatment solution having acomposition as shown in Example 4 and having a pH adjusted to 4.3.Immediately after the resulting mixture was treated at 37° C. for 30minutes, 10 mL of the treated solution was added to a bile treatmentsolution containing 1% of Oxgall. The mixture was treated at 37° C. for30 minutes and then, centrifuged at 5640×g for 10 minutes to recover thebacteria. The resulting bacteria were smeared on an agar plate. From theculture thus obtained, several single-colony derived strains wereselected, whereby B. breve YIT 4125 strain (which may hereinafter becalled “4125 strain”) and YIT 4126 strain (which may hereinafter becalled “4126 strain”) having improved tolerance to an acid and bile wereobtained.

By using these strains and the strain A, syrup-containing fermentedmilks were prepared in a similar manner to that described in thebeginning of Example 6. After storage at 10° C. for 7 days, the numberof surviving bacteria after the successive treatments with an acid andbile was determined in a similar manner to that employed in Example 4.

The drinking test was performed also on these samples. Ten healthy maleadult volunteers were divided into two groups and were asked to take,for 3 days, 100 mL/day of either one of a sample prepared using the 4125strain or the strain A. Feces on the next day after completion of thedrinking were collected and the extent of the recovery of theadministered bacteria was measured using a T-CBP plate. After aninterval of 1 week, the volunteers of each group were asked to take asample different from the first one and the number of bacteria recoveredwas counted. An average of the results of the measurements conductedtwice for each strain was designated as the number of bacteriarecovered.

Table 7 shows the results of a survival ratio after the successivetreatments with an acid and bile and results of recovery of bacteriaadministered through drinking, each on Day 7 after the storage of thefermented milk; and a change ratio in the number of living bacteria inthe fermented milk product during 7 day storage. The “change rate” asused herein means the number of living bacteria, in terms of logarithm,which decreased per day during low-temperature storage.

TABLE 7 Strain A 4125 Strain 4126 Strain Initial number of bacteria 3.7× 10⁸ 3.3 × 10⁸ 3.7 × 10⁸ The number of surviving 7.8 × 10⁵ 1.3 × 10⁸2.1 × 10⁸ bacteria after successive treatments Survival ratio 0.2% 39.4%56.8%The number of bacteria recovered

Strain A 4125 Strain 4126 Strain The number of bacteria recovered 6.47.0 Not test (logarithm)Change rate of the number of living bacteria in the product

Strain A 4125 Strain Change rate of the number of living bacteria perday 0.22 0.05

The 4125 and 4126 strains obtained in the present Example exhibitedtolerance of 20% or greater to an acid and bile. The number of bacteriarecovered when the 4125 strain was administered through drinking was1×10⁷ cfu/mL or greater.

A change rate of the number of living bacteria during the storage of theproduct was small, suggesting that the number of bacteria was kept high.

From the above-described results, it has been found that when thesurvival ratio after successive treatments with an acid and bile is 20%or greater, the number of bacteria recovered after administration ofthem through drinking becomes 10⁷ cfu/mL.

A storage test at 10° C. was then performed for three weeks on theabove-described samples. A survival ratio of bacteria in the productafter storage for 3 weeks is shown in Table 8.

TABLE 8 Strain A 4125 Strain Survival ratio after 3 weeks 21.0% 69.6%

A survival ratio of the bacteria in the product obtained using the 4125strain was high even after storage for 3 weeks.

Example 7

A commercially available product was next investigated. Tolerance to anacid and bile of each of a commercially-availableBifidobacterium-bacteria- and syrup-containing fermented milk (Fermentedmilk E) and the fermented milk of Example 6 prepared using the 4125strain (each fermented milk was stored for 7 days) was measured as inExample 4.

At the same time, one healthy adult volunteer was asked to take theseproducts after lunch at an interval of one week and the number ofBifidobacterium bacteria derived from each product and contained in thefeces on the next day was counted. The number of Bifidobacteriumbacteria derived from the product was calculated as follows: first byselectively breeding only Bifidobacterium bacteria in the feces on a TOSplate (prepared in a similar manner to the T-CBPC plate of Example 1except for the omission of an antibiotic), identifying, from them, theproduct-derived Bifidobacterium bacteria by Random Amplified PolymorphicDNA Fingerprinting (RAPD method) and calculating the number of recoveredbacteria based on their existing proportion. The results are shown inTable 9.

TABLE 9 Product Commercially available product E 4125 Strain The initialnumber of bacteria 5.2 × 10⁷ 1.7 × 10⁸ The number of surviving 8.5 × 10⁶7.3 × 10⁷ bacteria after successive treatments with an acid and bileSurvival ratio 16.3% 42.9%The number of bacteria recovered

Strain E 4125 Strain The number of bacteria recovered (logarithm) <6.897.20

With regards to the commercially available syrup-containing fermentedmilk, a survival ratio of bacteria after successive treatments with anacid and bile was less than 20% and the recovery ratio of bacteria afteradministration through drinking did not reach 1×10⁷ cfu/mL.

INDUSTRIAL APPLICABILITY

The present invention makes it possible to conveniently and accuratelyassess the extent to which Bifidobacterium bacteria in a fermented milkfood reach the intestines.

The present invention also makes it possible to provideBifidobacterium-bacteria-containing fermented milk food and drinkpermitting the high extent to which the bacteria reach the intestineseven after storage.

1. An isolated Bifidobacterium breve strain exhibiting viability of 20%or greater after treatment in an acid treatment solution at pH 4.3 at37° C. for 30 minutes, followed by treatment in a bile treatmentsolution containing 1% bile at 37° C. for 30 minutes compared to thesame strain prior to said acid and bile treatments.
 2. TheBifidobacterium breve strain of claim 1, which is Bifidobacterium breveYIT strain 4125 (FERM BP-7813).
 3. The Bifidobacterium breve strain ofclaim 1, which is Bifidobacterium breve YIT strain 4126 (FERM BP-7814).4. A fermented food or drink which has been fermented by, and contains,the Bifidobacterium breve strain of claim
 1. 5. The fermented food ordrink of claim 4 which is a fermented milk product.
 6. The fermentedfood or drink of claim 4 which contains saccharide-containing syrup. 7.A method for providing viable Bifidobacterium breve to the intestine ofa subject, comprising orally administering the food or drink of claim 4to said subject.
 8. The Bifidobacterium breve strain of claim 1 thatafter storage for 7 days at 10° C., and after oral ingestion by a human,is recovered from the human intestines at a titer of 1×10⁷ cfu/ml ormore.
 9. A method for making a Bifidobacterium breve strain that hasincreased viability after refrigerated storage comprising: treating aBifidobacterium breve strain in an acid treatment solution at pH 4.3 at37° C. for 30 minutes, treating said Bifidobacterium breve strain in abile treatment solution containing 1% bile at 37° C. for 30 minutes,isolating a single-colony derived Bifidobacterium breve strain thatmaintains a viability of 20% or more after treatment with acid and bileunder the conditions stated above, compared to the viability of the samestrain that has not been treated with bile and acid.